Mammalian sperm must undergo a final maturation, either in vivo or in vitro, before they can fertilize an oocyte. This maturation of sperm and the changes that occur in sperm are known as capacitation. The mechanisms of capacitation are not understood. It is necessary to first identify the mechanisms by which sperm capacitate in a defined system in vitro before any attempt can be made to understand in vivo events. Changes in the second messengers, cAMP, intracellular calcium (Cai) and intracellular pH(pHi) occur during capacitation. We propose to use image analysis techniques on individual sperm to determine the role and regulation of sperm Cai and pHi during capacitation. The model we will use is the bovine because of the abundance of sperm, sperm head shape that is easily imaged, capacitation conditions using heparin have been well worked out and inhibitors or non-capacitating conditions, in the absence of heparin or with glucose present, are also well known. Specific aim A is to determine the role of Cai i capitation by determining if an increase in Cai is needed to complete capacitation, if accelerating or delaying an increase in Cai will alter capacitation kinetics, if the increase in Cai is due to increase d influx of calcium, decreased efflux, decreased mitochondrial calcium uptake or a combination of these mechanisms as well as determining the importance of different mechanisms that regulate Cai homeostasis. To accomplish this, sperm will be loaded with the fluorescent calcium indicator Fura2 and Cai in the sperm head determined with quantitative image analysis. The Cai of capacitated or non-capacitated sperm which do or do not acrosome react in response to soluble zona proteins will be determined. The influx of calcium will be examined by the influx of Mn++ into Fura2 loaded sperm and the rate of Fura2 quench. The efflux of calcium from the cytoplasm will be examined by pulsing sperm with calcium from the caged calcium chelator NITR5. Specific Aim B is to determine the role of pHi in capacitation by determining if an increase in pHi is needed to complete capacitation and if increases in Cai stimulate the increase in pHi or the reverse. To accomplish this, sperm will be loaded with the fluorescent pH indicator BCECF and pHi in the sperm head determined with quantitative image analysis. The pHi of capacitated or non-capacitated sperm that do or do not acrosome react in response to soluble zona proteins will be determined. Mechanisms of pHi regulation in sperm will be examined using acid or alkaline loading of sperm and determining the rate of pHi recovery to resisting levels and how this is altered with capacitation. By inhibiting or stimulating specific pHi regulatory mechanisms it should be possible to determine their importance by whether capacitation kinetics can be changed.